Mechanism of inactivation of influenza viruses by
immobilized hydrophobic polycations
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Citation
Hsu, B. B. et al. “Mechanism of Inactivation of Influenza Viruses
by Immobilized Hydrophobic Polycations.” Proceedings of the
National Academy of Sciences 108.1 (2011) : 61-66. ©2011 by
the National Academy of Sciences.
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http://dx.doi.org/10.1073/pnas.1017012108
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National Academy of Sciences (U.S.)
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Final published version
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Thu May 26 06:31:48 EDT 2016
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http://hdl.handle.net/1721.1/64967
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Detailed Terms
Mechanism of inactivation of influenza viruses
by immobilized hydrophobic polycations
Bryan B. Hsua, Sze Yinn Wongb, Paula T. Hammondb, Jianzhu Chenc,d, and Alexander M. Klibanova,e,1
Departments of aChemistry, bChemical Engineering, and cBiology, and dKoch Institute for Integrative Cancer Research, and eDepartment of Biological
Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139
antiviral ∣ biocide ∣ virucidal coating
I
nfectious diseases, including influenza, kill millions of people a
year and sicken hundreds of millions (1, 2). In the United States
alone, there are tens of thousands of influenza-related deaths annually (3). The influenza virus’s propensity for genomic recombination and mutation (antigenic shift and drift, respectively)
can generate new strains to which humans have no previously
developed immunity—a common factor in influenza pandemics
(4, 5). A jarring example in modern history is the Spanish Flu of
1918–19, which killed up to 100 million people worldwide (6); the
H1N1 (“Swine Flu”) virus of 2009 has shown that such pandemics
remain an acute threat. Although vaccines and such antivirals as
zanamivir (Relenza®) and oseltamivir (Tamiflu®) are somewhat
effective, they have serious limitations: The former must predict
the next seasonal flu strain months in advance for current eggbased vaccines (7), while the latter are unreliable because of rapid
mutations in the influenza virus epitopes (8).
A complementary approach to vaccines and drugs is to inactivate the virus (or any pathogenic microbe) with biocides during its
transmission through inanimate objects, which is a major route
for nosocomial infections (9, 10). However, biocidal formulations
commonly applied as solutions can evaporate and be used up or
wiped away, making the efficacy of this approach dependent on
the frequency of its reapplication.
We have demonstrated (11) that coating (“painting”) with certain hydrophobic polycations renders surfaces permanently antibacterial and antifungal, retaining their disinfectant properties
even after multiple washes (12, 13). Recently, this antimicrobial
activity has been expanded to influenza viruses (14, 15). Herein
we mechanistically elucidate this phenomenon. Specifically, we
have found that upon contact with N;N-dodecyl,methyl-PEI coatings, aqueous solutions of influenza A viruses (including human
and avian, both wild-type and mutant zanamivir-resistant, strains)
are completely disinfected; this correlates with a disappearance
of viral proteins, although significant quantities of viral RNA
are still in solution. Based on this and other evidence, we conclude that these solutions are disinfected by the removal of viral
particles that irreversibly adhere to the hydrophobic polycationic
coatings; the latter then cause disintegration (including RNA
release) and inactivation of the adhered viruses.
methyl-PEI, we explored whether the nature of the underlying
solid object plays a role. Because the virucidal activity of these
hydrophobic polycations was previously discovered with coated
glass slides (14, 15), we added to these studies chemically unrelated polyethylene and polypropylene. As seen in Table 1, bare
(i.e., uncoated) slides of each material exhibited only partial
or no virucidal activity against a waterborne influenza virus. In
contrast, when coated with N;N-dodecyl,methyl-PEI, all three
types of surfaces completely disinfected the aqueous solutions
of the virus, indicating that this property is independent of the
surface treated.
While quantifying the influenza virus by the plaque assay (16)
allowed to titer the infectivity of solution, the fate of the viral
particles heretofore remained obscure. In particular, we could
not discriminate between the following possibilities: (i) The
viruses collide with the coated surface, undergo irreversible inactivation, and bounce off back into solution; or (ii) the viruses collide with the coated surface and irreversibly adhere to it in either
an infectious or noninfectious form. To distinguish between these
alternative scenarios, we employed the viral nucleoprotein (NP)
(a prevalent influenza protein, some 1,000 copies/viron) (4) as a
marker for the viral particles. Note that although NP is an internal viral protein, it can be easily quantified following lysis of viral
particles.
The ELISA data for NP—i.e., the viral particles—in aqueous
solutions of an influenza virus (WSN strain) incubated between
pairs of bare glass, polypropylene, or polyethylene slides revealed
marked loss (presumably due to nonspecific adsorption) of virons
for glass but little for the two polymers (Table 1). When the slides
were coated with N;N-dodecyl,methyl-PEI, however, the viral
particles completely disappeared from solution regardless of
the material coated (Table 1). All subsequent studies reported
herein were performed with polyethylene slides because they displayed far less background adsorption than glass and were easier
to work with than polypropylene slides.
To test the generality of the foregoing observations, we expanded our investigation to other influenza viruses, namely a
human pathogenic strain (PR8), as well as avian pathogenic
wild-type (TurkWt) and mutant zanamivir-resistant (TurkMu)
strains (Table 2). As seen in Fig. 1A, virucidal activities of bare
polyethylene slides are negligible against all of these strains. In
contrast, the N;N-dodecyl,methyl-PEI-coated slides are completely virucidal (Fig. 1A), thus indicating that the hydrophobic polycationic coating is able to disinfect aqueous solutions of a variety
of diverse influenza strains. Separately, the independent ELISA
results depicted in Fig. 1B show that while the bare slides had
minor effect on viral particle concentrations, the viral particles
disappeared from solution after incubation with the polycaAuthor contributions: B.B.H., P.T.H., J.C., and A.M.K. designed research; B.B.H. and S.Y.W.
performed research; B.B.H., S.Y.W., J.C., and A.M.K. analyzed data; and B.B.H. and A.M.K.
wrote the paper.
The authors declare no conflict of interest.
Results and Discussion
Prior to embarking on a mechanistic investigation of virucidal
properties of surfaces coated (“painted”) with N;N-dodecyl,
www.pnas.org/cgi/doi/10.1073/pnas.1017012108
1
To whom correspondence should be addressed. E-mail: klibanov@mit.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1017012108/-/DCSupplemental.
PNAS ∣ January 4, 2011 ∣ vol. 108 ∣ no. 1 ∣ 61–66
CHEMISTRY
N,N-Dodecyl,methyl-polyethylenimine coatings applied to solid
surfaces have been shown by us to disinfect aqueous solutions
of influenza viruses. Herein we elucidate the mechanism of this
phenomenon. Infectivity-, protein-, RNA-, and scanning electron
microscopy-based experiments reveal that, upon contact with
the hydrophobic polycationic coating, influenza viruses (including
pathogenic human and avian, both wild-type and drug-resistant,
strains) irreversibly adhere to it, followed by structural damage
and inactivation; subsequently, viral RNA is released into solution,
while proteins remain adsorbed.
APPLIED BIOLOGICAL
SCIENCES
Contributed by Alexander M. Klibanov, November 15, 2010 (sent for review October 8, 2010)
Table 1. Effect of glass, polypropylene, and polyethylene slides coated with N − N-dodecyl,methyl-PEI on WSN influenza strain’s viral
infectivity and concentration of viral particles in solution
Infectivity (by plaque assay)*
Surface
Glass
Polypropylene
Polyethylene
Initially
20.4 ± 2.1
20.4 ± 2.1
20.4 ± 2.1
After contact
with bare slides
10.9 ± 0.5
18.1 ± 1.9
15.1 ± 3.2
Viral particles (by ELISA)*,
After contact with
polycation-coated slides
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
Initially
21.0 ± 2.2
21.0 ± 2.2
21.0 ± 2.2
After contact with
bare slides
1.4 ± 2.1
17.8 ± 1.1
17.1 ± 1.2
†
After contact with
polycation-coated slides
0.0 ± 1.6
−1.0 ± 2.2
0.1 ± 1.2
*Values represent titers: ðmean std devÞ × 104 pfu∕mL
†
Assessed by measuring the concentration of viral nucleoprotein (NP); the high loss of viral particles for bare glass slides is likely due to nonspecific adsorption
of exogenous viral NP
tion-coated slides. Thus using influenza’s NP as a marker reveals
a strong correlation between the disinfection of the viral solution
and the disappearance of the viral particles from it, suggesting
that the influenza viral particles adhere to the hydrophobic polycationic coatings. Note that this adherence takes place regardless
of variables arising from the source of culture, suspension media,
and antigenicity associated with the different strains used
(Table 2).
To independently verify these results and to test for other influenza viral proteins in solution, we examined N;N-dodecyl,
methyl-PEI-coated slides against a solution of the purified
PR8H virus (which differs from PR8L only by its much higher
titer; Table 2) and confirmed its complete disinfection (Fig. 2A).
Additionally, we analyzed the initial viral solution, as well as that
after exposure to either bare or polycation-coated slides, by
means of SDS/PAGE. Even a visual inspection of protein band
profiles in lanes 1, 2, and 3 in Fig. 2B reveals some loss for each
protein after the exposure, with by far the most dramatic one
occurring with the virus incubated with the coated slides (lane
3 in Fig. 2B). Quantification of these observations by gel-scanning
densitometry indicates a loss of 94% of viral protein after incubation with the coated slides versus only a 34% loss after incubation with control, bare slides (Fig. 2C). [That the bare slides
demonstrated the same losses in virucidal activity (Fig. 2A) as
in viral protein point to a nonspecific removal of the virus from
solution.] The observed complete loss of virucidal activity and a
concurrent nearly complete loss of viral protein suggest that a
small fraction of inactivated influenza virus and/or proteincontaining fragments thereof remains in solution even following
incubation with coated slides, likely due to the much higher viral
titer required for SDS/PAGE used in these experiments.
These findings support the second aforementioned scenario,
namely that the viral particles irreversibly adhere to the N;Ndodecyl,methyl-PEI-coated surface, thereby vanishing from solution. To mechanistically explore this process further, we endeavored to assess the infectivity of the influenza viruses adhered
to the coated surface. Because such viruses are not amenable
to cell-based infectivity assays, we examined instead how much
viral RNA could be detected in solution. Its substantial presence
there would evidence the inactivation of the surface-bound
viruses because they obviously cannot be infective without their
RNA. To this end, we used real-time reverse-transcriptase PCR
(qRT–PCR) with the probe and primers detecting influenza
virus’s seventh RNA segment; i.e., the genomic region encoding
the matrix (M 1 ) protein (4, 17). Using the same influenza strains
(Table 2), negligible losses of viral RNA from solution were
observed in the case of bare polyethylene slides (Fig. 3). Importantly, and in sharp contrast to the results from our proteinbased assays (Fig. 2), however, all viral solutions incubated with
the N;N-dodecyl,methyl-PEI-coated slides exhibited significant
quantities of viral RNA. This leakage of viral RNA from the
surface-adhered viral particles points to a profound damage to
the virus and its inevitable demise.
To ensure that the RNA detected in solution was indeed leaked
from the interior of the viral particle, we pretreated all viral
samples with the ribonuclease enzymes to digest any exogenous
single- or double-stranded RNA that may be present (18–20).
Control experiments confirmed that this enzymatic pretreatment
completely digests RNA and that addition of ribonuclease’s
inhibitor prevents subsequent RNA digestion (Fig. S1). There
was also no nonspecific amplification from N;N-dodecyl,methylPEI putatively leached into solution (Fig. S2). A comparison of
genomic sequences between influenza viral samples before and
after contact with the coating revealed at least a 95% sequence
identity (less than a 100% identity is likely due to inherent errors
in the sequencing technology) (21). All these results support the
conclusion that the detected viral RNA is, in fact, endogenous
viral genomic material leaked into solution after contact with
the hydrophobic polycationic coating.
The recovery of RNA from the immobilized-N;N-dodecyl,
methyl-PEI-contacted viral solutions was about half of that from
the uncoated slides. This contrasts to our protein studies that
have thus far suggested an all-or-none virus inactivation scenario.
Because the root of this partial recovery of genomic material may
point to an additional mechanism in the polycation’s virucidal activity, we directly visualized the viral particles on the surfaces
using scanning electron microscopy (SEM). Micrographs of the
WSN strain of influenza virus on a plain silicon wafer showed
no visible damage to the viral structure (Fig. 4A). In contrast, exposure of the viruses to the coated surface revealed a mixture of
two extremes: either a substantial structural damage leaving a
gaping hole (Fig. 4B) or no noticeable effect (Fig. 4C). Specifically, out of 132 exposed viral particles surveyed, 54% exhibited
the type of structural damage seen in Fig. 4B. Interestingly, this
Table 2. Strains of influenza A viruses used in this study and their properties
Short name
WSN
PR8H
PR8L†
TurkWt
TurkMu
Influenza A viral strain
Initial titer (pfu∕mL)*
WSN/33 (H1N1)
PR/8/34 (H1N1)
PR/8/34 (H1N1)
turkey/MN/833/80 (H4N2)
turkey/MN/833/80 (H4N2)
4.5ð0.6Þ × 106
1.3ð0.5Þ × 108
0.9ð0.2Þ × 106
1.0ð0.1Þ × 106
4.3ð0.3Þ × 106
Culture conditions;
suspension conditions
Tissue; DME-HEPES/10% FBS
Egg; HEPES/saline
Egg; HEPES/PBS/saline
Egg; allantoic fluid
Egg; allantoic fluid
Notes
Laboratory strain, nonpathogenic
Human pathogenic; sucrose-gradient purified
Human pathogenic; sucrose-gradient purified
Avian pathogenic
Avian pathogenic; zanamivir-resistant
*Determined via plaque assay after ribonuclease pretreatment; the titer is that of a 10-μL droplet incubated with slides prior to a 100-fold dilution upon
washing. See Materials and Methods for details.
†
An approximately 100-fold dilution of PR8H with PBS
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Hsu et al.
B
NP
B
A
APPLIED BIOLOGICAL
SCIENCES
A
CHEMISTRY
Fig. 1. The effect of polyethylene slides coated with N;N-dodecyl,methyl-PEI
challenged with aqueous solutions of various influenza A viruses. See Table 2
for viral strain abbreviations and details (note that “H” and “L” after PR8
stand for high and low titer of the virus, respectively). (A) Virucidal activity
of bare (white bars) and polycation-coated polyethylene slides (hatchmarked bars) against various influenza virus strains. (B) Relative quantities
of viral nucleoprotein (NP) disappeared from solution after incubation with
bare (gray bars) and with polycation-coated polyethylene slides (black bars).
C
value agrees with the fraction of RNA recovered from the coated
slides as compared to the bare slides (Fig. 3). As to the observed
(Fig. 4 B and C) bimodal distribution, note that even viral particles that appear intact may be actually damaged on the side
facing the N;N-dodecyl,methyl-PEI coating hidden from view;
this type of damage would also likely hinder escape of viral RNA
into solution.
One might have expected that no RNA should be recovered in
solution at all due to nonspecific interactions between the negatively charged nucleic acid and the surface-situated hydrophobic
polycation molecules if not for the fact that the derivatization
of PEI with long alkyl moieties sterically interferes with such
interactions (22–24). Note in this regard that elemental analysis
of N;N-dodecyl,methyl-PEI used herein shows approximately
1.6 dodecyl moieties per monomer unit, corresponding to, on
average, 60% of monomers in PEI doubly dodecylated and
40% singularly dodecylated, which is considerably greater than
in the previous reports (22–24).
Finally, we tested whether a monomeric structural analog
of N;N-dodecyl,methyl-PEI, namely dodecyltrimethylammonium
bromide (DTAB), was also capable of inactivating the influenza
virus. To this end, we challenged the influenza virus with serial
dilutions of DTAB for the latter’s concentration required for
50% inhibition (IC50 ) (25) (and also its toxic concentration
TC50 —i.e., that reducing MDCK cell viability by 50%). The
DTAB’s IC50 value was found to be 21 5 μg∕mL [which was
fourfold lower than its 84 26 μg∕mL TC50 value, in agreement
with literature (26)]. Therefore, the antiviral activity observed
with our surface-immobilized hydrophobic polycations appears
to be inherent to the quaternary ammonium monomeric unit. Indeed, a similar quaternary ammonium salt, benzalkonium chloride, is also capable of inactivating influenza virus (27–29).
Hsu et al.
Fig. 2. The effect of polyethylene slides coated with N;N-dodecyl,methyl-PEI
on high-titer human pathogenic PR8 influenza virus strain (PR8H, Table 2).
(A) Virucidal activity of PR8H by bare (white bars) and N;N-dodecyl,
methyl-PEI-coated polyethylene slides (hatch-marked bars). (B) SDS/PAGE
of the concentrated PR8H strain run on a 7% polyacrylamide gel and stained
with the commassie blue dye. Lanes 1, 2, and 3 represent virus samples prior
to assay, after contact with bare slides and after contact with polycationcoated slides, respectively. (C) Protein disappeared from solution by bare
slides (gray bars) and polycation-coated slides (black bars), as determined
by gel-scanning densitometry.
Based on the experimental evidence obtained herein, we propose a framework for influenza virus inactivation depicted in
Fig. 5. When viral particles strike the N;N-dodecyl,methyl-PEIcoated surface due to thermal motion (Fig. 5A), they can adhere
to it (Fig. 5B) through hydrophobic and electrostatic interactions
(11). As a result of such interactions, virus’s disintegration ensues
manifesting itself in a leakage of RNA into solution and a loss of
infectivity (Fig. 5C). In this regard it is instructive to note that,
upon interactions with polycations, model lipid vesicles can undergo lateral segregation and flip-flopping (30) of phospholipids,
PNAS ∣
January 4, 2011 ∣ vol. 108 ∣ no. 1 ∣
63
N;N-Dodecyl,methyl-PEI was applied from its 50 mg∕mL solution in CHCl3
by painting slides with a 3∕8 inch nylon-bristled paint brush (Loew–Cornell)
and dried in a chemical hood; this painting procedure was repeated twice
more. The square 2.5-cm slides were cleaned by sonication in isopropyl alcohol for 5 min and autoclaved prior to use. Glass slides were cut from standard
microscope slides (VWR International), while low-density polyethylene and
polypropylene slides were cut from 12“ × 12“ × 1∕16“ sheets (McMaster–Carr
Supply Co.).
Fig. 3. Relative quantities of influenza A viral RNA in solution prior to assay
(white bars), after incubation with bare polyethylene slides (hatch-marked
bars), and after incubation with N;N-dodecyl,methyl-PEI-coated polyethylene
slides (cross-hatched bars).
ultimately fluidizing their membrane (31). These types of effects
would disrupt the ordered lipid rafts (32, 33) and leaflet asymmetry (34) in influenza viral envelopes.
Conclusions
Through investigation into how aqueous solutions of various human and avian influenza viruses are disinfected by N;N-dodecyl,
methyl-PEI coatings, we found that this phenomenon was dependent on neither the nature of the surface that was coated nor
the strain of influenza A virus. A direct correlation was found
between the exposed solution’s losses of infectivity and of viral
proteins, thereby indicating that the viral particles adhere to
the surface-immobilized hydrophobic polycation. Additional analysis of the disinfected solution revealed significant quantities of
RNA therein that stemmed from the viral particles, thereby evidencing that the integrity of the latter was compromised allowing
the release of their genomic material. This conclusion was independently verified by direct SEM observations. A monomeric
analog of N;N-dodecyl,methyl-PEI, DTAB, was also found to
inactivate the influenza virus, suggesting that the antiviral activity
is inherent to the hydrophobic quaternary ammonium salt moiety,
which is retained in even a polymeric and surface-immobilized
form.
Materials and Methods
Polymer Synthesis and Immobilization. Unless otherwise noted, all chemicals
were from Sigma-Aldrich Chemical Co. N;N-Dodecyl,methyl-PEI was synthesized as described previously (14, 16, 23). Briefly, linear 217-kDa PEI prepared
by hydrolysis of 500-kDa poly(2-ethyl-2-oxazoline) in concentrated HCl at 125
°C (23) was dodecylated, followed either by methylation with CH3 I to give the
final quaternary ammonium polycation or by dialysis within a cellulose membrane (3.5-kDa cutoff) against hexane. Elemental analysis of N-dodecyl-PEI
performed by Columbia Analytical Labs yielded a C∶H∶N of 72.3∶12.8∶4.0
by weight (or 21.1∶44.5∶1.0 by mol).
A
B
C
Fig. 4. Scanning electron microscopy (SEM) images of the WSN strain of influenza virus after exposure to plain (A) and N;N-dodecyl,methyl-PEI-coated
(B and C) silicon wafers. Of the latter, a larger fraction of viral particles
showed substantial structural damage (B), while a smaller fraction showed
no visible damage (C); see text for details. (Scale bars, 100 nm.)
64 ∣
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Virus Culture and Ribonuclease Pretreatment. The influenza A/WSN/33 (H1N1)
virus was cultured from MDCK cells (14). Sucrose-gradient purified influenza
A/PR/8/34 (H1N1) virus suspended in HEPES-saline buffer was used as obtained from Charles River Labs. Wild-type and zanamivir-resistant influenza
A/MN/833/80 (H4N2) viruses were prepared from egg allantoic fluid (15).
Exogenous viral RNA (single- and double-stranded) was digested in all viral samples by incubating 100 μL of an influenza virus solution with 15 μL of
ribonuclease III buffer, 50 μL of ribonuclease III enzyme (1 U∕μL, Ambion), and
5 μL of 1,000-fold-diluted ribonuclease A (3;500 U∕μL, Invitrogen) in PBS for
1 h at 37 °C (18–20). Subsequently, 5 μL of Antiribonuclease (20 U∕μL, Ambion) was added, and the solution was incubated at RT for 15 min before
being placed on ice.
A 10-μL droplet of a viral aqueous solution was sandwiched between two
slides (glass, polyethylene, or polypropylene) in a 6 × 1.5 cm polystyrene Petri
dish. After a 5-min incubation at RT, the slides were separated and washed
with 990 μL of PBS. Viral samples were collected prior to contact with slides,
after contact with bare slides, or after contact with polycation-coated
slides (16).
Plaque assays were used to quantify the infectious viral particles (in pfu/
mL) in solution by infecting a monolayer of MDCK cells in six-well plates with
200 μL of a viral solution (16).
ELISA. An ELISA of influenza virus NP was performed with a 96-well maxisorp
immunoplate (Nunc Intl.): the wells were incubated with 100 μL of a 1∶1;000
dilution of goat anti-influenza A polyclonal antibody (AB1074, Millipore) in
PBS at 4 °C overnight and then washed thrice with 100 μL of a solution of
0.2% (w∕v) I-block (Applied Biosystems) and 0.05% Tween 20 (v∕v) in PBS
(PBST), followed by a further incubation with an additional 100 μL of PBST
for 3 h at RT. Then 100-μL volumes of viral samples, prepared by combining
equal volumes of virus solution and 2X PBST, were added. After incubation
overnight at 4 °C, the wells were washed thrice with 100 μL of PBST and incubated with 100 μL of a 3∶10;000 dilution of mouse anti-influenza A biotinylated monoclonal antibody (Millipore) in PBST for 3 h at RT. This was
followed by washing thrice with 100 μL of PBST, incubation with 100 μL of
a 1∶200 dilution of a streptavidin-horseradish peroxidase conjugate (Millipore) in PBS for 2 h at RT and washing thrice with 100 μL of PBST. Colorimetric
development of 50 μL of 1-Step Ultra TMB (Thermo Scientific) at RT was
stopped with 50 μL of 0.2 M H2 SO4 after incubation for 12, 6, 9, or 4 min
with WSN, PR8L, TurkWt, or TurkMu influenza viruses, respectively. Light absorbances measured at 450 nm for virus samples prior to assay, as well as after
contact with bare or N;N-dodecyl,methyl-PEI-coated slides, were referenced
to standard curves constructed from serially diluted stock samples and normalized to their plaque-assay determined titers.
SDS/PAGE. Sucrose-gradient purified PR8H viral samples were disrupted
with 1% SDS in PBS such that the final concentration was 0.1% SDS. Proteins
from viral samples were concentrated and washed using Amicon Ultra 0.5-mL
microcentrifuge filters (10-kDa cutoff, Millipore), according to the manufacturer’s instructions. Briefly, proteins were concentrated at RT by centrifugation at 14;000 × g and then washed thrice with 400 μL of 0.1% SDS in PBS by
additional centrifugation. The final 14.0 μL of washed and concentrated protein solution was run on a SDS/PAGE using a 7% Tris-acetate NuPAGE gel (Invitrogen) and Tris-acetate buffer kit (Invitrogen) according to manufacturer’s
instructions. Briefly, 14.0 μL of concentrated protein was combined with
5.4 μL of 4X lithium dodecylsulfate sample buffer, and 2.2 μL of 10X reducing
agent prior to incubation at 70 °C for 10 min. The prepared samples and a
boiled protein ladder (10–250 kDa, New England BioLabs) were added to
the gel and subjected to electrophoresis for 60 min at 150 V. The resultant
gel was stained with SimplyBlue comassie blue stain (Invitrogen) and imaged
on an Alpha Imager 2200 (Cell Biosciences). Gel-scanning densitometry analysis was performed using ImageJ software (NIST) by calibrating the measured pixel density to optical density and then developing a standard
curve with a power-law fit using the protein bands of serially diluted viral
samples to which assay samples were referenced. Assignment of protein
bands was based on the literature data (4, 35).
Hsu et al.
Fig. 5. Proposed mechanism of influenza virus inactivation by N;N-dodecyl,methyl-PEI coatings. (A) An influenza viral particle diffuses to the polycationcoated surface from solution and (B) adheres to it. Significant damage imparted by the immobilized hydrophobic polycations is then incurred by the adhered
viral particle such that (C) its genomic material (RNA) leaks into solution leaving the viral particle inactivated on the surface.
Scanning Electron Microscopy. Plain and N;N-dodecyl,methyl-PEI-coated 1-cm
square silicon wafers were placed in a Petri dish, and a 10-μl droplet of an
influenza virus solution was placed in the center before sandwiching with
a plain silicon wafer to spread the droplet. This system was incubated at
RT for 30 min before fixing the samples with Karnovsky’s fixative kit (Polysciences). To this end, the samples were incubated in the fixing solution (2%
paraformaldehyde and 2.5% glutaraldehyde in 0.1 M Na phosphate buffer)
for 2 h and rinsed for 10 min in the fresh buffer. The samples were then incubated in the dark in a 1% osmium tetroxide solution for 1 h before sequentially rinsing in 35%, 50%, 70%, 95%, and 100% aqueous ethanol for 10 min
each, followed by dehydration thrice in 100% ethanol. The samples were
then freeze-dried in liquid nitrogen and sputter-coated before imaging with
a JEOL JSM-6700F SEM instrument at a 100,000X magnification.
Detergent Studies. The viability of MDCK cells after a 1-h incubation at
RT with 100 μL of serially diluted DTAB in PBS was determined using the
CellTiter96 Aqueous Nonradioactive Cell Proliferation Assay (Promega), an
MTS-based assay for mitochondrial activity (36), and was performed according to manufacturer’s instructions. The measured 50% toxicity concentration
ACKNOWLEDGMENTS. We thank Jayanta Haldar for providing the avian influenza A virus strains used herein; Peter Tieu for experimental assistance; and
Liguo Wu, Koushik Mukherjee, Mishtu Dey, Peter Goldman, Eugene Antipov,
Laura Jennings, Alyssa Larson, and Ching-Hung Shen for insightful discussions. This work was supported by the U.S. Army through the Institute of
Soldier Nanotechnologies at the Massachusetts Institute of Technology under
contract DAAD-19-02-D0002 with the Army Research Office.
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PNAS ∣
January 4, 2011 ∣ vol. 108 ∣ no. 1 ∣
65
APPLIED BIOLOGICAL
SCIENCES
RT-PCR and Sequencing. Aliquots of 200 μL of WSN virus strain samples prior
to assay and after contact with N;N-dodecyl,methyl-PEI-coated slides underwent RNA extraction as described above. Reverse transcription was performed using the Omniscript RT Kit (Qiagen) according to manufacturer’s
instructions with Antiribonuclease (20 U∕μL, Ambion AM2690) as the ribonuclease inhibitor with the aforementioned primers (IDT) (17). For amplification
of the newly transcribed viral DNA, the TopTaq DNA Polymerase (Qiagen)
with a dNTP mix (Promega) and primers were used according to manufacturer’s instructions. Sequencing was performed by the MIT Biopolymers
Facility.
(TC50 ) of DTAB was calculated using replicates of dose-response fitted curves
(Origin Labs OriginPro v8.1).
The 50% inhibitory concentration (IC50 ) of DTAB against PR8 was measured by incubating serial dilutions of detergent with PR8 for 30 min at
RT prior to determination of their infectivity by plaque assay (16, 25). Plaques
were easily identifiable at and below 43 μg∕mL DTAB, while above this level
(e.g., at 85 μg∕mL) cells were opaque, thus making potential plaques indistinguishable. Hence the IC50 value was calculated using replicates of doseresponse fitted curves (Origin Labs OriginPro v8.1) to DTAB concentrations
not exceeding 43 μg∕mL.
CHEMISTRY
Viral RNA Extraction and Quantification with qRT-PCR. RNA was extracted from
200 μL of a viral solution using the PureLink viral RNA/DNA kit (Invitrogen)
according to manufacturer’s instructions giving a final 110 μL of viral RNA,
stored at −80 °C. Quantification was performed with the RNA Ultrasense
qRT-PCR mix (Invitrogen) according to manufacturer’s instructions, with
the primers and probe (IDT) sequences used identical to those previously
developed (17). Real-time reverse-transcriptase PCR was performed on an
Applied Biosystems 7500 real-time PCR system as follows: 50 °C for 15 min,
95 °C for 2 min, and then 50 cycles of 95 °C for 15 sec and 60 °C for
32 sec. RNA samples from virus prior to assay, after contact with bare slides,
and after contact with polycation-coated slides were referenced to standard
curves constructed from serially diluted stock samples and normalized to
plaque-assay titers (Applied Biosystems 7500 Software v2.0.1). All samples
and standard curves were included on the same reaction plate.
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